ISSN 00062979, Biochemistry (Moscow), 2013, Vol. 78, No. 11, pp. 12931297. © Pleiades Publishing, Ltd., 2013. Original Russian Text © N. I. Fedotcheva, E. N. Mokhova, 2013, published in Biokhimiya, 2013, Vol. 78, No. 11, pp. 16431648.
Mitochondrial Models of Pathologies with Oxidative Stress. Efficiency of Alkalization to Reduce Mitochondrial Damage N. I. Fedotcheva1,2 and E. N. Mokhova2,3* 1
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, 142290 Pushchino, Moscow Region, Russia; fax: (4967) 330553; Email: [email protected] 2 Lomonosov Moscow State University, Research Institute of Mitoengineering, 119991 Moscow, Russia; fax: (495) 9395945 3 Lomonosov Moscow State University, Belozersky Institute of PhysicoChemical Biology, 119991 Moscow, Russia; fax: (495) 9393181; Email: [email protected] Received July 31, 2013 Revision received August 8, 2013 Abstract—Previously, we developed a method to monitor the development of oxidative stress in isolated liver mitochondria. The method is based on recording of membrane potential changes in response to sequential introduction of low concentra tions (520 μM) of tertbutyl hydroperoxide (tBHP). It allows monitoring of the extent of amplification or attenuation of oxidative stress caused by external influences (changes in incubation conditions, additions of biologically active substances). Based on this method, we created a mitochondrial model for the study and improvement of treatment of pathologies asso ciated with oxidative stress. The following two processes were simulated in the experiments: 1) introduction of desferal for treatment of serious diseases caused by cell overload with iron (high desferal concentrations were shown to suppress mito chondrial energetics); 2) efficiency of alkalization to reduce mitochondrial damage induced by oxidative stress. The exper iments have shown that even a small increase in pH (alkalization) increases the amount of tBHP that can be added to mito chondria before the MPTP (“mitochondrial permeability transition pore”) is induced. The effect of alkalization was shown to be close to the effect of cyclosporin A in the pH range 7.27.8. The mechanism of the similarities of these effects in the organism and in mitochondrial suspensions is explained by the increase in toxic reactive oxygen species in both systems under oxidative stress. DOI: 10.1134/S0006297913110102 Key words: oxidative stress, mitochondria, tertbutyl hydroperoxide, cyclosporin Asensitive pore, desferal, alkalization
Oxidative stress causes molecular and cellular dam age leading to diverse serious diseases, although cells pos sess various mechanisms for regulation of mitochondrial redox status [1]. Oxidative stress often results from changes in the external environment, e.g. oxygen concen tration [2]. Oxidative stressinduced cell damage is often caused by the opening of the nonspecific Ca2+dependent mito chondrial permeability transition pore in the inner mito chondrial membrane (MPTP). It was discovered over 30 years ago by Hunter and Haworth [3]. This significant discovery was appreciated only after MPTP opening had been shown to cause cell death. Abbreviations: DSF, desferal; MPTP, mitochondrial permeabil ity transition pore; tBHP, tertbutyl hydroperoxide; Δψ, trans membrane difference of electrical potentials on the inner mito chondrial membrane. * To whom correspondence should be addressed.
Verseci et al. suggested that superoxide radical and hydrogen peroxide, which are known to accumulate in mitochondria, exit from mitochondria into the incuba tion medium. H2O2 can interact with Fe2+, facilitating the formation of highly toxic hydroxyl radical, which can also have toxic effects from the outer medium [4, 5]. As high catalase activity complicated the experi ments with hydrogen peroxide on mitochondria isolated from liver and some other tissues, we started using tert butyl hydroperoxide (tBHP) instead of hydrogen perox ide ([6, 7] and references therein). tBHP is similar to hydrogen peroxide in many respects, including its effects on mitochondria. Monitoring of mitochondrial redox status and its changes in the course of medical treatment is often used to identify the risk of the development of diseases and/or efficiency of the treatment of the pathology. Determination of the ratio of reduced and oxidized glutathione (GSH and GSSG) is often used in disease
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diagnosis, because GSH is one of the major components of the antioxidant system in animal tissues including those of humans [8]. Such measurements, as well as determination of tBHP toxicity (when its concentration increases in some samples) are very time consuming. We developed earlier a rapid method for monitoring cellular redox status following the dynamics of mitochon drial membrane potential changes (Δψ) caused by intro duction of successive additions of low tBHP concentra tions [9]. This approach allows study of induction of oxidative stress in intact mitochondria, which have the highest Δψ. Antioxidant and prooxidant activities of bio logically active compounds can be studied by quickly varying different parameters in the course of experiments. The character of responses to small additions shows that tBHP is transported electrophoretically into mito chondria. Once this transport ended, the Δψ returns to baseline. Such membrane potential dynamics provides a quick answer to the question about the tested compound (or different conditions of mitochondria incubation) enhancing or inhibiting the onset of oxidative stress. Earlier experiments showed this transport to proceed at high speed already with the addition of 2 μM Fe2+ into the incubation medium [9]. Similar transport occurs also without any added iron, i.e. endogenous iron is enough for its manifestation. We used this method for the development of liver mitochondriabased mitochondrial models of patholo gies caused by defects in mitochondrial energetics. In this work, we have studied the following two processes. The first includes introduction of desferal for the treatment of pathologies resulting from cell overload with iron. Many publications by Verseci et al. are dedicat ed to the thorough study of the MPTP stimulated by oxidative stress in the presence of iron. In particular, they studied MPTP formation caused by administration of a large excess of iron into isolated liver mitochondria [10]. A series of severe pathologies resulting from iron excess have been described, many of them remaining incurable even today. When the state of the patient deteriorates, dif ferent methods of reduction of iron content in blood are used, including bloodletting. An injection of an iron specific reagent, desferal, is an alternative. Desferal does not enter the bloodstream when administered orally. Details and references can be found in [11]. We have also studied the second mitochondrialevel process associated with the method proposed by Edgar Cayce (USA). This is the method of nonpharmaceutical treatment of patholo gies caused by excessive pH decrease in body fluids by restoration of acid–base balance in body fluids (blood, lymph, saliva, etc.), shifting it in the “alkaline” direction by selecting special diets for different pathologies [12]. Cayce has published many books on the recom mended methods of treatment. Efficiency of many of his recommendations has been already confirmed in medical research.
Our experiments have shown a slight increase in the incubation medium pH to increase dramatically the amount of tBHP that must be added to liver mitochon dria to open the MPTP. Thus, we have confirmed effi ciency of alkalization on the model of liver mitochondria suspension.
MATERIALS AND METHODS Male Wistar rats from Institute of Theoretical and Experimental Biophysics vivarium were used in the experiments. All the used reagents were produced by Sigma (USA). Liver mitochondria were isolated by the differential centrifugation method in medium containing 300 mM sucrose, 10 mM Tris, pH 7.4. Mitochondria were isolated by a twostage centrifugation without washing; the isola tion medium contained no EDTA or EGTA. The value of Δψ was recorded using a TPP+selective electrode with subsequent computer processing. Mitochondria were incubated in hypotonic sucrose medi um containing 75 mM sucrose, 10 mM Mops, pH 7.4 (pH was adjusted by KOH), 1.5 mM phosphate, 5 mM KCl. In some experiments salt incubation medium con taining 120 mM KCl, 10 mM Hepes, pH 7.25, 1.5 mM phosphate, 0.5 mM MgCl2 was used, with TPP+ added to concentration 1 μM. Concentration of mitochondrial protein in the measuring cell was 1 mg/ml in all the sam ples. Details of the described experimental conditions are provided in the legends to the corresponding figures. Spectral analysis of the formation of desferal com plexes with iron (FeSO4) was performed using an Ocean Optics USB4000 spectrophotometer. Complex formation was accompanied by the appearance of brown color at 435nm wavelength. The peak amplitude at this wave length depends on the concentration of each of the com plex components [13]. The spectra were recorded in medium containing 75 mM sucrose, 10 mM Mops, pH 7.4.
RESULTS Figure 1a shows data on the effects of desferal (DSF) on the development of oxidative stress obtained using our method. One can see that Δψ changes caused by succes sive tBHP additions (at final concentration of 20 μM each) in the presence of 200 μM DSF (curve 2) differ only slightly from the control (curve 1), although a minor prooxidant effect of DSF can be observed. However, addition of 500 μM DSF to mitochondria causes a distinct prooxidant effect (Fig. 1a, curve 3). This amount of DSF is close to the amount used in medical treatment. EDTA (50 μM) restores Δψ decreased in the presence of tBHP and DSF. BIOCHEMISTRY (Moscow) Vol. 78 No. 11 2013
MITOCHONDRIAL MODELS OF PATHOLOGIES WITH OXIDATIVE STRESS
DISCUSSION Experiments with desferal have shown iron (Fe2+) to bind with EDTA far better than with DSF. It should be added that (Fe2+) affinity to EDTA is several orders of magnitude higher than its affinity to DSF. Although EDTA also has a high affinity to aluminum, in our con ditions EDTA can be considered as a specific iron chela tor. EDTA can be used to treat pathologies with exces sive iron accumulation, first in model experiments with animals. It is likely to bind iron in the bloodstream, thereby reducing its entry into cells. The most interest ing result of this study was obtained in the experiments carried out to test the efficiency of alkalization for treat ing several pathologies caused by oxidative stress. We showed that a slight increase in the pH of the incubation BIOCHEMISTRY (Moscow) Vol. 78 No. 11 2013
a
1.2 TPP+ concentration, μM
EDTA ↓ 3
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0.5 DSF + Fe ± EDTA 0.4 Optical density
It should be noted that EDTA can lower the concen tration of reduced iron more efficiently and with no known side effects, as indicated by the spectral data pre sented in Fig. 1b. DSF forms a browncolored complex with iron hav ing an absorption maximum at 435 nm. Figure 1b shows the spectra of desferal added to incubation medium with out mitochondria in the presence of 50 (curve 1) and 100 μM (curve 2) FeSO4. The next curves (3 and 4) show the spectra of DSF complex with iron formed in EDTA containing medium with the same iron concentrations. Figure 1b shows that EDTA, an iron chelator, significant ly reduces the absorption of the DSF complex with iron due to partial binding of Fe2+. This method can be used to estimate the amount of DSFbound iron under different incubation conditions as well as in the presence of biolog ically active compounds. Figure 2a shows the effect of pH of the mitochondr ial incubation medium on tBHPinduced MPTP open ing. It shows mitochondrial membrane potential changes after successive tBHP additions at final concen tration 10 μM each at different values of the pH of the medium. It can be seen that increasing the pH causes the number of additions leading to MPTP opening to increase manyfold. At pH of about 7.8 the number of tBHP additions is almost the same as when adding cyclosporin A. Figure 2b shows the data from experiments where the same method was used to pass the entire studied pH range three times in increments of 0.1 pH unit. This fig ure shows mitochondrial resistance to tBHP increases dramatically when approaching pH 7.8. It is also important to note that the number of tBHP additions in the presence of previously introduced cyclosporin A (2 μM) at any given pH value to be the same (data not shown), and its effect on the number of tBHP additions was similar to the effect of medium alka lization to pH 7.8.
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Wavelength, nm Fig. 1. Effect of DSF on tBHPinduced MPTP opening. a) Changes in membrane potential caused by successive tBHP addi tions (at final concentration 20 μM each) in the control (1) and in the presence of 200 (2) and 500 μM (3) desferal. Addition of 50 μM EDTA restores the membrane potential. Incubation medium: 75 mM sucrose, 10 mM MOPS, 1.5 mM phosphate, 5 mM KCl, pH 7.4, 4 mM pyruvate. b) Absorption spectra (with 435nm maximum) of DSF (100 μM) complex with iron at con centration 50 (1) and 100 μM (2); effect of 50 μM EDTA on the formation of DSF complex with iron at FeSO4 concentration of 50 (3) and 100 μM (4). Incubation medium is the same, but with out substrate and phosphate.
medium increases dramatically the amount of tBHP that can be added before opening of the MPTP when added to incubation medium with isolated liver mitochondria. At the same time, the amount of tBHP added to mito chondria without MPTP opening under the same condi tions but in the presence of cyclosporin A was practical
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b
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Fig. 2. Effect of pH of the incubation medium on tBHPinduced MPTP opening. a) Original curves of mitochondrial membrane potential changes caused by successive tBHP additions at final concentration of 10 μM each at different pH values of the incubation medium and in the presence of cyclosporin A (Cs). b) Dependence of tBHP concentration required for MPTP opening on pH of the incubation medium. Incubation medium: 75 mM sucrose, 10 mM MOPS, 1.5 mM phosphate, 5 mM KCl, 4 mM pyruvate.
ly the same. Thus, slight alkalization of incubation medi um has no effect on tBHP transport, but it delays MPTP opening. This conclusion causes absolutely no doubts on the model of liver mitochondria suspension. In all the numerous samples, pH increase of at least onetenth of a pH unit and more caused both Δψ increase and increase in the number of tBHP additions before pore opening. A previously described [9] method of nonparametric statis tics, “rule of signs”, shows very high probability of our conclusions being true, if the sign of the effect is repeated in all seven of the conducted independent experiments (see tables in the description of this method in [14]). Experiments described in this paper are much more reli able as not only the general direction of the effects, but also their values are similar. All these data show the mitochondrial pathology model developed in our studies to be useful for solving a variety of medical and biological problems. This is due to the combination of its following features: 1) MPTP prop erties depend on many conditions. The data published in [14] clearly illustrate that not only changes in MPTP activators, but also increased incubation time with them change the properties of the pore (even if pH of the incu bation medium does not change), in particular, its sensi tivity to cyclosporin A. In our experiments, MPTP open ing resulted from the increase in prooxidant tBHP con centration, and it depended only on tBHP concentra tion; 2) experiments were carried out on mitochondria with the highest Δψ, i.e. on intact mitochondria. Lipophilic cations of TPP+type are known to accumu late electrophoretically, i.e. they are concentrated in mitochondria with the highest Δψ. A more detailed dis cussion of this property and relevant references can be found in the section “Some Methodological Aspects of
Experiments with Mitochondria…” in [15]; 3) mito chondria were incubated under conditions close to phys iological. The pH of the incubation medium was ~7.4, which is close to the average pH of blood. In the main experiments, mitochondria were isolated without wash ing and addition of such stabilizers as EGTA, EDTA, BSA, and others. E. Cayce stated that a small increase in pH (alkaliza tion) of body fluids will provide a cure for a number of serious pathologies (it could be measured by pH changes in blood, lymph, urine, saliva [12]). He suggested meas uring saliva pH with test strips. The mitochondrial model used in our experiments enables further research in this direction. For example, it seems interesting and impor tant to understand the mechanism of the strong effect of a small increase in pH on MPTP induction by the proox idant tBHP. The methods developed by E. Cayce are described not only in many of his own publications, but also in a number of articles published by doctors, healers, and patients working together with him in his institute (USA). We have chosen to refer to the publication that is the most accessible for the study and application of his methods [12]. We express heartfelt gratitude to Vladimir P. Skulachev and Innocent V. Skulachev for various help during the research. REFERENCES 1. 2.
Kowaltowski, A. J., and Vercesi, A. E. (1999) Free Radic. Biol. Med., 26, 463471. Papa, S., and Skulachev, V. P. (1997) Mol. Cell Biochem., 174, 305319. BIOCHEMISTRY (Moscow) Vol. 78 No. 11 2013
MITOCHONDRIAL MODELS OF PATHOLOGIES WITH OXIDATIVE STRESS 3. 4. 5.
6.
7.
8.
Haworth, R. A., and Hunter, D. R. (2000) J. Bioenerg. Biomembr., 32, 9196. Kowaltowski, A. J., Netto, L. E., and Vercesi, A. E. (1998) J. Biol. Chem., 273, 1276612769. Vercesi, A. E., Kowaltowski, A. J., Grijalba, M. T., Meinicke, A. R., and Castilho, R. F. (1997) Biosci Rep., 17, 4352. Cervinkova, Z., Krivakova, P., Labajova, A., Rousar, T., Lotkova, H., Kucera, O., Endlicher, R., Cervinka, M., and Drahota, Z. (2009) Arch. Toxicol., 83, 6372. Endlicher, R., Krivakova, P., Rauchova, H., Nuskova, H., Cervinkova, Z., and Drahota, Z. (2009) Physiol. Res., 58, 685692. Cao, L., Waldon, D., Teffera, Y., Roberts, J., Wells, M., Langley, M., and Zhao, Z. (2013) Anal. Bioanal. Chem., 405, 26352642.
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9. Fedotcheva, N. I., and Mokhova, E. N. (2013) Biochemistry (Moscow), 78, 7579. 10. Pardo Andreu, G. L., Inada, N. M., Vercesi, A. E., and Curti, C. (2009) Arch. Toxicol., 83, 4753. 11. Devanur, L. D., Evans, R. W., Evans, P. J., and Hider, R. C. (2008) Biochem. J., 409, 439447. 12. Bolton, B. (compiler) (2004) An Edgar Cayce Encyclopedia of Foods for Health and Healing (McCarey, W. A., ed.), A.R.E. Press, Virginia Beach, Virginia. 13. Fernandez, V., and Winkelmann, G. (2005) Biometals, 18, 5362. 14. Sigel, S. (1956) Nonparametric Statistics in Science and Behavior, N.Y. 15. Broekemeier, K. M., and Pfeiffer, D. R. (1995) Biochemistry, 34, 1644016449. 16. Mokhova, E. N. (2012) Biophys. Rev. Lett., 7, 113.
Mitochondrial Models of Pathologies with Oxidative Stress. Efficiency of Alkalization to Reduce Mitochondrial Damage N. I. Fedotcheva1,2 and E. N. Mokhova2,3* 1
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, ul. Institutskaya 3, 142290 Pushchino, Moscow Region, Russia; fax: (4967) 330553; Email: [email protected] 2 Lomonosov Moscow State University, Research Institute of Mitoengineering, 119991 Moscow, Russia; fax: (495) 9395945 3 Lomonosov Moscow State University, Belozersky Institute of PhysicoChemical Biology, 119991 Moscow, Russia; fax: (495) 9393181; Email: [email protected] Received July 31, 2013 Revision received August 8, 2013 Abstract—Previously, we developed a method to monitor the development of oxidative stress in isolated liver mitochondria. The method is based on recording of membrane potential changes in response to sequential introduction of low concentra tions (520 μM) of tertbutyl hydroperoxide (tBHP). It allows monitoring of the extent of amplification or attenuation of oxidative stress caused by external influences (changes in incubation conditions, additions of biologically active substances). Based on this method, we created a mitochondrial model for the study and improvement of treatment of pathologies asso ciated with oxidative stress. The following two processes were simulated in the experiments: 1) introduction of desferal for treatment of serious diseases caused by cell overload with iron (high desferal concentrations were shown to suppress mito chondrial energetics); 2) efficiency of alkalization to reduce mitochondrial damage induced by oxidative stress. The exper iments have shown that even a small increase in pH (alkalization) increases the amount of tBHP that can be added to mito chondria before the MPTP (“mitochondrial permeability transition pore”) is induced. The effect of alkalization was shown to be close to the effect of cyclosporin A in the pH range 7.27.8. The mechanism of the similarities of these effects in the organism and in mitochondrial suspensions is explained by the increase in toxic reactive oxygen species in both systems under oxidative stress. DOI: 10.1134/S0006297913110102 Key words: oxidative stress, mitochondria, tertbutyl hydroperoxide, cyclosporin Asensitive pore, desferal, alkalization
Oxidative stress causes molecular and cellular dam age leading to diverse serious diseases, although cells pos sess various mechanisms for regulation of mitochondrial redox status [1]. Oxidative stress often results from changes in the external environment, e.g. oxygen concen tration [2]. Oxidative stressinduced cell damage is often caused by the opening of the nonspecific Ca2+dependent mito chondrial permeability transition pore in the inner mito chondrial membrane (MPTP). It was discovered over 30 years ago by Hunter and Haworth [3]. This significant discovery was appreciated only after MPTP opening had been shown to cause cell death. Abbreviations: DSF, desferal; MPTP, mitochondrial permeabil ity transition pore; tBHP, tertbutyl hydroperoxide; Δψ, trans membrane difference of electrical potentials on the inner mito chondrial membrane. * To whom correspondence should be addressed.
Verseci et al. suggested that superoxide radical and hydrogen peroxide, which are known to accumulate in mitochondria, exit from mitochondria into the incuba tion medium. H2O2 can interact with Fe2+, facilitating the formation of highly toxic hydroxyl radical, which can also have toxic effects from the outer medium [4, 5]. As high catalase activity complicated the experi ments with hydrogen peroxide on mitochondria isolated from liver and some other tissues, we started using tert butyl hydroperoxide (tBHP) instead of hydrogen perox ide ([6, 7] and references therein). tBHP is similar to hydrogen peroxide in many respects, including its effects on mitochondria. Monitoring of mitochondrial redox status and its changes in the course of medical treatment is often used to identify the risk of the development of diseases and/or efficiency of the treatment of the pathology. Determination of the ratio of reduced and oxidized glutathione (GSH and GSSG) is often used in disease
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diagnosis, because GSH is one of the major components of the antioxidant system in animal tissues including those of humans [8]. Such measurements, as well as determination of tBHP toxicity (when its concentration increases in some samples) are very time consuming. We developed earlier a rapid method for monitoring cellular redox status following the dynamics of mitochon drial membrane potential changes (Δψ) caused by intro duction of successive additions of low tBHP concentra tions [9]. This approach allows study of induction of oxidative stress in intact mitochondria, which have the highest Δψ. Antioxidant and prooxidant activities of bio logically active compounds can be studied by quickly varying different parameters in the course of experiments. The character of responses to small additions shows that tBHP is transported electrophoretically into mito chondria. Once this transport ended, the Δψ returns to baseline. Such membrane potential dynamics provides a quick answer to the question about the tested compound (or different conditions of mitochondria incubation) enhancing or inhibiting the onset of oxidative stress. Earlier experiments showed this transport to proceed at high speed already with the addition of 2 μM Fe2+ into the incubation medium [9]. Similar transport occurs also without any added iron, i.e. endogenous iron is enough for its manifestation. We used this method for the development of liver mitochondriabased mitochondrial models of patholo gies caused by defects in mitochondrial energetics. In this work, we have studied the following two processes. The first includes introduction of desferal for the treatment of pathologies resulting from cell overload with iron. Many publications by Verseci et al. are dedicat ed to the thorough study of the MPTP stimulated by oxidative stress in the presence of iron. In particular, they studied MPTP formation caused by administration of a large excess of iron into isolated liver mitochondria [10]. A series of severe pathologies resulting from iron excess have been described, many of them remaining incurable even today. When the state of the patient deteriorates, dif ferent methods of reduction of iron content in blood are used, including bloodletting. An injection of an iron specific reagent, desferal, is an alternative. Desferal does not enter the bloodstream when administered orally. Details and references can be found in [11]. We have also studied the second mitochondrialevel process associated with the method proposed by Edgar Cayce (USA). This is the method of nonpharmaceutical treatment of patholo gies caused by excessive pH decrease in body fluids by restoration of acid–base balance in body fluids (blood, lymph, saliva, etc.), shifting it in the “alkaline” direction by selecting special diets for different pathologies [12]. Cayce has published many books on the recom mended methods of treatment. Efficiency of many of his recommendations has been already confirmed in medical research.
Our experiments have shown a slight increase in the incubation medium pH to increase dramatically the amount of tBHP that must be added to liver mitochon dria to open the MPTP. Thus, we have confirmed effi ciency of alkalization on the model of liver mitochondria suspension.
MATERIALS AND METHODS Male Wistar rats from Institute of Theoretical and Experimental Biophysics vivarium were used in the experiments. All the used reagents were produced by Sigma (USA). Liver mitochondria were isolated by the differential centrifugation method in medium containing 300 mM sucrose, 10 mM Tris, pH 7.4. Mitochondria were isolated by a twostage centrifugation without washing; the isola tion medium contained no EDTA or EGTA. The value of Δψ was recorded using a TPP+selective electrode with subsequent computer processing. Mitochondria were incubated in hypotonic sucrose medi um containing 75 mM sucrose, 10 mM Mops, pH 7.4 (pH was adjusted by KOH), 1.5 mM phosphate, 5 mM KCl. In some experiments salt incubation medium con taining 120 mM KCl, 10 mM Hepes, pH 7.25, 1.5 mM phosphate, 0.5 mM MgCl2 was used, with TPP+ added to concentration 1 μM. Concentration of mitochondrial protein in the measuring cell was 1 mg/ml in all the sam ples. Details of the described experimental conditions are provided in the legends to the corresponding figures. Spectral analysis of the formation of desferal com plexes with iron (FeSO4) was performed using an Ocean Optics USB4000 spectrophotometer. Complex formation was accompanied by the appearance of brown color at 435nm wavelength. The peak amplitude at this wave length depends on the concentration of each of the com plex components [13]. The spectra were recorded in medium containing 75 mM sucrose, 10 mM Mops, pH 7.4.
RESULTS Figure 1a shows data on the effects of desferal (DSF) on the development of oxidative stress obtained using our method. One can see that Δψ changes caused by succes sive tBHP additions (at final concentration of 20 μM each) in the presence of 200 μM DSF (curve 2) differ only slightly from the control (curve 1), although a minor prooxidant effect of DSF can be observed. However, addition of 500 μM DSF to mitochondria causes a distinct prooxidant effect (Fig. 1a, curve 3). This amount of DSF is close to the amount used in medical treatment. EDTA (50 μM) restores Δψ decreased in the presence of tBHP and DSF. BIOCHEMISTRY (Moscow) Vol. 78 No. 11 2013
MITOCHONDRIAL MODELS OF PATHOLOGIES WITH OXIDATIVE STRESS
DISCUSSION Experiments with desferal have shown iron (Fe2+) to bind with EDTA far better than with DSF. It should be added that (Fe2+) affinity to EDTA is several orders of magnitude higher than its affinity to DSF. Although EDTA also has a high affinity to aluminum, in our con ditions EDTA can be considered as a specific iron chela tor. EDTA can be used to treat pathologies with exces sive iron accumulation, first in model experiments with animals. It is likely to bind iron in the bloodstream, thereby reducing its entry into cells. The most interest ing result of this study was obtained in the experiments carried out to test the efficiency of alkalization for treat ing several pathologies caused by oxidative stress. We showed that a slight increase in the pH of the incubation BIOCHEMISTRY (Moscow) Vol. 78 No. 11 2013
a
1.2 TPP+ concentration, μM
EDTA ↓ 3
1.0
2
0.8
1
tBHP 20 μM each DSF ↓ ↓
0.6 0.4 0.2
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100
200
300
400
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0.5 DSF + Fe ± EDTA 0.4 Optical density
It should be noted that EDTA can lower the concen tration of reduced iron more efficiently and with no known side effects, as indicated by the spectral data pre sented in Fig. 1b. DSF forms a browncolored complex with iron hav ing an absorption maximum at 435 nm. Figure 1b shows the spectra of desferal added to incubation medium with out mitochondria in the presence of 50 (curve 1) and 100 μM (curve 2) FeSO4. The next curves (3 and 4) show the spectra of DSF complex with iron formed in EDTA containing medium with the same iron concentrations. Figure 1b shows that EDTA, an iron chelator, significant ly reduces the absorption of the DSF complex with iron due to partial binding of Fe2+. This method can be used to estimate the amount of DSFbound iron under different incubation conditions as well as in the presence of biolog ically active compounds. Figure 2a shows the effect of pH of the mitochondr ial incubation medium on tBHPinduced MPTP open ing. It shows mitochondrial membrane potential changes after successive tBHP additions at final concen tration 10 μM each at different values of the pH of the medium. It can be seen that increasing the pH causes the number of additions leading to MPTP opening to increase manyfold. At pH of about 7.8 the number of tBHP additions is almost the same as when adding cyclosporin A. Figure 2b shows the data from experiments where the same method was used to pass the entire studied pH range three times in increments of 0.1 pH unit. This fig ure shows mitochondrial resistance to tBHP increases dramatically when approaching pH 7.8. It is also important to note that the number of tBHP additions in the presence of previously introduced cyclosporin A (2 μM) at any given pH value to be the same (data not shown), and its effect on the number of tBHP additions was similar to the effect of medium alka lization to pH 7.8.
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Wavelength, nm Fig. 1. Effect of DSF on tBHPinduced MPTP opening. a) Changes in membrane potential caused by successive tBHP addi tions (at final concentration 20 μM each) in the control (1) and in the presence of 200 (2) and 500 μM (3) desferal. Addition of 50 μM EDTA restores the membrane potential. Incubation medium: 75 mM sucrose, 10 mM MOPS, 1.5 mM phosphate, 5 mM KCl, pH 7.4, 4 mM pyruvate. b) Absorption spectra (with 435nm maximum) of DSF (100 μM) complex with iron at con centration 50 (1) and 100 μM (2); effect of 50 μM EDTA on the formation of DSF complex with iron at FeSO4 concentration of 50 (3) and 100 μM (4). Incubation medium is the same, but with out substrate and phosphate.
medium increases dramatically the amount of tBHP that can be added before opening of the MPTP when added to incubation medium with isolated liver mitochondria. At the same time, the amount of tBHP added to mito chondria without MPTP opening under the same condi tions but in the presence of cyclosporin A was practical
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b
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Fig. 2. Effect of pH of the incubation medium on tBHPinduced MPTP opening. a) Original curves of mitochondrial membrane potential changes caused by successive tBHP additions at final concentration of 10 μM each at different pH values of the incubation medium and in the presence of cyclosporin A (Cs). b) Dependence of tBHP concentration required for MPTP opening on pH of the incubation medium. Incubation medium: 75 mM sucrose, 10 mM MOPS, 1.5 mM phosphate, 5 mM KCl, 4 mM pyruvate.
ly the same. Thus, slight alkalization of incubation medi um has no effect on tBHP transport, but it delays MPTP opening. This conclusion causes absolutely no doubts on the model of liver mitochondria suspension. In all the numerous samples, pH increase of at least onetenth of a pH unit and more caused both Δψ increase and increase in the number of tBHP additions before pore opening. A previously described [9] method of nonparametric statis tics, “rule of signs”, shows very high probability of our conclusions being true, if the sign of the effect is repeated in all seven of the conducted independent experiments (see tables in the description of this method in [14]). Experiments described in this paper are much more reli able as not only the general direction of the effects, but also their values are similar. All these data show the mitochondrial pathology model developed in our studies to be useful for solving a variety of medical and biological problems. This is due to the combination of its following features: 1) MPTP prop erties depend on many conditions. The data published in [14] clearly illustrate that not only changes in MPTP activators, but also increased incubation time with them change the properties of the pore (even if pH of the incu bation medium does not change), in particular, its sensi tivity to cyclosporin A. In our experiments, MPTP open ing resulted from the increase in prooxidant tBHP con centration, and it depended only on tBHP concentra tion; 2) experiments were carried out on mitochondria with the highest Δψ, i.e. on intact mitochondria. Lipophilic cations of TPP+type are known to accumu late electrophoretically, i.e. they are concentrated in mitochondria with the highest Δψ. A more detailed dis cussion of this property and relevant references can be found in the section “Some Methodological Aspects of
Experiments with Mitochondria…” in [15]; 3) mito chondria were incubated under conditions close to phys iological. The pH of the incubation medium was ~7.4, which is close to the average pH of blood. In the main experiments, mitochondria were isolated without wash ing and addition of such stabilizers as EGTA, EDTA, BSA, and others. E. Cayce stated that a small increase in pH (alkaliza tion) of body fluids will provide a cure for a number of serious pathologies (it could be measured by pH changes in blood, lymph, urine, saliva [12]). He suggested meas uring saliva pH with test strips. The mitochondrial model used in our experiments enables further research in this direction. For example, it seems interesting and impor tant to understand the mechanism of the strong effect of a small increase in pH on MPTP induction by the proox idant tBHP. The methods developed by E. Cayce are described not only in many of his own publications, but also in a number of articles published by doctors, healers, and patients working together with him in his institute (USA). We have chosen to refer to the publication that is the most accessible for the study and application of his methods [12]. We express heartfelt gratitude to Vladimir P. Skulachev and Innocent V. Skulachev for various help during the research. REFERENCES 1. 2.
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